Adipose tissue extrinsic factor: Obesity-induced inflammation and the role of the visceral lymph node

Accepted Manuscript Adipose tissue extrinsic factor: Obesity-induced inflammation and the role of the visceral lymph node

Aaron M. Magnuson, Josephine K. Fouts, Daniel P. Regan, Andrea D. Booth, Steve W. Dow, Michelle T. Foster PII: DOI: Reference:

S0031-9384(18)30105-7 doi:10.1016/j.physbeh.2018.02.044 PHB 12104

To appear in:

Physiology & Behavior

Received date: Revised date: Accepted date:

25 August 2017 24 December 2017 22 February 2018

Please cite this article as: Aaron M. Magnuson, Josephine K. Fouts, Daniel P. Regan, Andrea D. Booth, Steve W. Dow, Michelle T. Foster , Adipose tissue extrinsic factor: Obesity-induced inflammation and the role of the visceral lymph node. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phb(2017), doi:10.1016/j.physbeh.2018.02.044

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Adipose Tissue Extrinsic Factor: Obesity-Induced Inflammation and the Role of the Visceral Lymph Node

Aaron M. Magnuson1, Josephine K. Fouts1, Daniel P. Regan2, Andrea D. Booth1, Steve W.

IP

T

Dow2 and Michelle T. Foster1

CR

Department of Food Science and Human Nutrition1, Department of Microbiology, Immunology

US

and Pathology2, Colorado State University, Fort Collins, CO 80523, USA

AN

Running Head: Intra-Abdominal Immunity in Metabolic Disease

To whom correspondence should be addressed:

M

Dr. Michelle T. Foster, PhD.

1571 Campus Delivery

CE

500 West Lake Street

PT

Colorado State University

ED

Department of Food Science and Human Nutrition

Fort Collins, CO 80523

AC

Phone: (970) 491-6189 Fax: (970) 491-3875

E-mail: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT Obesity-related adverse health consequences occur predominately in individuals with upper body fat distribution commonly associated with increased central adiposity. Visceral adipose tissue accumulation is described to be the greatest driver of obesity-induced inflammation, however evidence also supports that the intestines fundamentally contribute to the development of obesity-

IP

T

induced metabolic disease. The visceral adipose depot shares the same vasculature and lymph

CR

drainage as the small intestine. We hypothesize that the visceral lymph node, which drains adipose tissue and the gastrointestinal tract, is central to the exacerbation of systemic pro-

US

inflammation. Male C57BL/6 mice were fed CHOW or high fat diet (HFD) for 7 weeks. At termination the mesenteric depot, visceral lymph node and ileum, jejunum and Peyer’s patches

AN

were collected. Cytokine concentration was determined in adipose tissue whereas immune cell populations where investigated in the visceral lymph node and intestinal segments by flow

M

cytometry. Visceral adipose tissue and the gastrointestinal tract mutually influence immune cells

ED

enclosed within the visceral lymph node. HFD increased visceral lymph node immune cell number. This likely resulted from 1.) an increase in immune cells migration from the small

PT

intestines likely from activated dendritic cells that travel to the lymph node and 2.) cytokine effluent

CE

from visceral adipose tissue that promoted expansion, survival and retention of pro-inflammatory immune cells. Overall, the visceral lymph node, the immune nexus of visceral adipose tissue and

AC

the small intestines, likely plays a fundamental role in exacerbation of systemic pro-inflammation by HFD-induced obesity. The research of Tim Bartness greatly enhanced the understanding of adipose tissue regulation. Studies from his laboratory significantly contributed to our awareness of extrinsic factors that influence body fatness levels. Specifically, the work he produced eloquently demonstrated that adipose tissue was more complex than an insulating storage center; it was connected to our brains via the sympathetic and sensory nervous system. Mapping studies demonstrated that adipose tissue both receives and sends information to the brain. Further, his

ACCEPTED MANUSCRIPT lab demonstrated that nervous system connections contributed to lipolysis, thermogenesis and adipocyte proliferation and growth. The work of Tim Bartness will continue to influence adipose tissue research. As such, Tim Bartness directly inspired the following research. Adipose tissue extrinsic factors are not limited to the peripheral nervous system. The lymphatic system is an additional extrinsic factor that cross talks with adipose tissue, however its role in this context is

IP

T

under emphasized. Here we begin to elucidate how the lymphatic system may contribute to the

CR

comorbidities associated with visceral adipose tissue accumulation.

US

Keywords: Lymphatics, Pro-Inflammation, Central Obesity, Metabolic Disease, Visceral

AC

CE

PT

ED

M

AN

Adiposity

ACCEPTED MANUSCRIPT INTRODUCTION Obesity increases the risk of disease comorbidities including, but not limited to, cardiovascular disease [1], liver inflammation [2], insulin resistance and type-2-diabetes [3]. Systemic low-grade chronic inflammation is commonly described to drive tissue impairments that exacerbate disease risks [4-8]. Excessive adipose tissue accumulation in obesity is associated

IP

T

with regional inflammation, which subsequently drives systemic inflammation that contributes to

CR

deleterious health outcomes [9-12]. In particular, evidence supports that obesity-related adverse health consequences occur predominately in individuals with upper body fat distribution

US

commonly associated with increased central adiposity [5, 13-15]. Here we question; why is visceral adiposity so detrimental?

AN

Adipose tissue can be modified by both intrinsic and extrinsic factors. One extrinsic factor capable of adipose tissue cross talk is the lymphatic system. This association is particularly

M

important in inflammatory processes. Specifically, lymph nodes are predominantly embedded in

ED

adipose tissue [16], and are the primary site for the development of regional protective immune responses. Here lymph nodes serve as conduits that facilitate interactions between immune cells

PT

(e.g. monocytes, macrophages, dendritic cells, neutrophils and lymphocytes) and antigens. The

CE

visceral lymph node located within the intra-abdominal cavity collects effluent from the visceral adipose depot. In adipose tissue, immune cells within the lymph nodes serve as the responders

AC

to tissue injury or pathogen invasion and are fundamental for the development of protective immune responses. These responses are orchestrated by the production and release of both proand anti-inflammatory cytokines. Hence, lymph nodes serve as posts that continuously survey and monitor exposure of adipose tissue to potentially harmful pathogens and metabolites [17, 18]. Immune cells within lymph nodes can be recruited and activated to defend adipose tissue against tissue damage, toxicity or impaired function [19]. In turn, research demonstrates adipose tissue serves as an energy reservoir to support immune functions, creating a reciprocal relation where enhanced pro-inflammation drives adipocyte proliferation [20]. The link between obesity and

ACCEPTED MANUSCRIPT impaired immune function has been known for some time, yet there is very little understanding of how excessive adipose tissue deposition alters the lymphatic system. Thus far, excessive adiposity associated with obesity is a risk for immune detriments including, but not limited to, accelerated thymic aging, a reduction in vaccine efficacy and increased morbidity associated with pathogen infection [21-23]. We hypothesize that chronic adipose tissue-driven inflammation

IP

T

influences lymphatic tissue function, in part, by changes in resident immune cell populations. This,

CR

in turn, can affect systemic immunity and heighten disease risk. Obesity-mediated inflammation is therefore a disease state that involves adaptations in the extrinsic lymphatic system.

US

Human and rodent studies suggest the greatest driver of obesity-induced inflammation is visceral adipose tissue accumulation. As it accumulates and dysregulates during obesity, free

AN

fatty acids, adipokines and pro-inflammatory cytokines increase in circulation subsequently effecting insulin sensitive tissues such as the liver [24, 25]. However, the visceral adipose depot

M

shares the same vasculature and lymph drainage as the small intestine. Hence, evidence also

ED

supports that the intestines fundamentally contribute to the development of obesity-induced metabolic disease [26] by way of inflammation.

PT

The gastrointestinal (GI) tract is the first line of protection from antigens and

CE

immunomodulatory agents consumed from our diet or localized within our gut. The GI tract must continuously distinguish beneficial nutrients and commensal bacteria from harmful pathogens [27,

AC

28]. Maintenance of immunologic defense and intestinal homeostasis engages ~70% of the body’s immune cells [29], hence the intestine has considerable influence on systemic immune function. Consequently, the gastrointestinal immune system within the small intestine must maintain physiological gut homeostasis despite continual exposure to varying lumen contents. Within the small intestine, this is accomplished by a semi-hierarchical organization of immune regions. Immune response can occur at the epithelial surface of the lumen and lamina propria, where regional immune cells attempt to eliminate noxious pathogens instantaneously (For review see:[30]). These same immune cells also release signaling molecules that can prompt activation

ACCEPTED MANUSCRIPT of a greater immune response. Gut-associated lymphoid tissue (GALT), a network of lymphoid tissue containing immune cells (for review see: [31, 32]), also defends against pathogens. GALT associated immune cells play a critical role in the protection from and inhibition against the penetration of gut-derived pathogens. Overall, gut immune homeostasis is fundamental to the protection of systemic immunity.

IP

T

Much like adipose tissue, a diet high in fat can cause inflammation of the gastrointestinal

CR

tract. Acute or chronic treatment/intake of lipids can shift small intestine immunity toward proinflammation, this is best demonstrated in rodent models. For example, high fat diet (HFD) feeding

US

increases tumor necrosis factor α (TNFα) expression and nuclear factor κβ (NF- κβ) activation, markers of pro-inflammation, in the small intestines of mice [33]. Consistent with this others

AN

demonstrate that HFD increases TNFα concentration within the lamina propria of the small intestines as well as other pro-inflammatory markers such as interleukin 6 and 17 (IL6 and 17)

M

[34]. Epithelial cells also release IL6, as well as growth-related oncogene/cytokine-induced

ED

neutrophil chemoattractant-1 (GRO/CINC-1; stimulates locomotion and activation of neutrophils) in the presence of long chain fatty acids [35]. HFD-induced pro-inflammation in the small intestine

PT

likely results from epithelial cell injury, damaged morphology, that occurs as lipid transverses the

CE

enterocyte from the lumen [36]. Disruption of small intestine homeostasis by lipids causes epithelial and stroma cells of the small intestines to engage and activate lymphoid cells in attempt

AC

to sustain immunity, mediate inflammation and eventually incite resolution [37]. Overall, chronic inflammation, as occurs with intake of saturated fat diets, disrupts the mucosal barrier enhancing permeability of pathogens, which subsequently exacerbates pro-inflammation [38, 39]. Small intestines pro-inflammation, ensuing from acute or chronic exposure to HFD, influences systemic physiological and pathological processes. We postulate that both visceral adipose tissue accumulation and the gastrointestinal tract influence systemic immunity during obesity. Both tissues are likely responsible for the comorbidities associated with obesity. We hypothesize that the visceral lymph node, which drains

ACCEPTED MANUSCRIPT adipose tissue and the gastrointestinal tract, is central to the exacerbation of systemic proinflammation. A limitation of previous studies in this area include specific focus on one distinct gastrointestinal region. To examine comprehensively HFD-induced alterations of the small intestines requires distinction between different regions contained within. There are inherent anatomical and physiological differences between the duodenum, jejunum, and ileum (For review

IP

T

see:[30]). Primary differences that may influence immune regulation include differing populations of epithelial cells and absorption capacity. Therefore, characteristics of immunity may be specific

CR

to anatomical location among the gut. In addition, Peyers’s patches, organized lymphoid nodules

US

located throughout the jejunum and ileum, play a fundamental role in the maintenance of intestinal immune homeostasis, yet the role of these nodules is often underemphasized. In this study, we

AN

used a Westernized diet to induce obesity in mice to investigate if diet-induced obesity differentially changed immune cell populations in the Peyer’s patches and distinct segments of

M

the small intestines. Along with this we describe immune cell changes in the visceral lymph node,

ED

a structure proposed to control immigration of immune cells from the gut and enhance tolerance to gut derived pathogens [40]. Last, adipose tissue cytokine concentration was used to determine

Animals

AC

METHODS

CE

PT

if visceral lymph node immune cell alterations are related to visceral adipose tissue signaling.

Male C57BL/6 mice (n=10, aged 2-3 months, 27.7±0.22 g) from Jackson Laboratory, Bar Habor, Maine, were single housed under controlled conditions (12:12 light-dark cycle, 50–60% humidity, 25°C) and allowed one week of acclimation before experiment start. Following acclimation, mice were given free access to water and a standard CHOW or Westernized high fat and sugar diet (HFD) until termination at 7 weeks. Standard CHOW diet was (CHOW: Harlan Teklad LM485, Madison, WI.) 3.1 kcal/g with ~17% kcal from fat (~5.8% by weight of diet), ~25% protein, and

ACCEPTED MANUSCRIPT ~58% carbohydrate. The fats in the diet consisted of mono- and poly-unsaturated and saturated fatty acids (1.3%, 2.9% and 0.8% by weight of diet respectively). Westernized high fat and sugar diet (HFD) (Harlan Teklad, TD.08811, Madison, WI) was 4.7 kcal/g with ~45% kcal from fat (~23% by weight of diet, main source milkfat), ~14.7% protein and ~40.7% carbohydrate (34% sucrose). The fats in the diet consisted of mono- and poly-unsaturated and saturated fatty acids (31%, 8%

IP

T

and 61% respectively). Body mass and food intake were recorded weekly. Procedures were

CR

reviewed and approved by the Colorado State University Institutional Animal Care and Use

US

Committee.

Terminal Procedures (Blood Collection and Tissue Harvesting)

AN

Termination occurred after mice were on respective diets for 7 weeks. Mice were anesthetized with isoflurane and terminated by decapitation. Adipose depots were collected and weighed and

M

the mesenteric (visceral) depot was stored at -80°C. The visceral lymph node embedded inside

ED

the visceral depot was collected. Peyer’s patches (n = 5 mice, 4 collected Peyer’s Patches/mouse) were first collected from the small intestines (along the ileum and jejunum). Next 30mm portions

PT

of ileum and jejunum (n = 5 each), without visible Peyer’s patch portions, were collected. Intestine

CE

tissues and the visceral lymph node were placed into Hank’s balanced salt solution (HBSS) until

AC

further processed.

Adipose Tissue Cytokines Adipose tissue accumulation, especially visceral (central) adiposity, is associated with increases in cytokines that contribute to pro-inflammation. Cytokine concentration was measured in the visceral depot to associate adipose depot changes with extrinsic lymph node immune cell signaling. Interleukin (IL) proteins were analyzed because of their role in regulating immune response. In particular, these ILs include proteins which generally contribute to pro-inflammation, IL-1β and IL-6, and those that are demonstrated to be higher in lean animals, IL-5 and IL-13.

ACCEPTED MANUSCRIPT Visceral adipose tissue Interleukin -1β, -5, -6 and -13, as well as KC GRO (a.k.a. chemokine (CX-C motif) ligand 1 (CXCL1) and tumor necrosis factor α (TNFα) protein concentration was measured by Mesoscale (Rockville, Maryland). Two kits were utilized, a V-PLEX Proinflammatory Panel 1 Mouse Kit (Mesoscale; Cat No. K15048D-1, Rockville, Maryland) and a UPLEX customized IL-13 Mouse Kit (Mesoscale; Rockville, Maryland). Briefly, adipose tissues was

IP

T

placed in 2 ml tubes in HBSS+5% FBS (1mL), minced using scissors and sonicated on ice. Tissue

CR

homogenates were then treated according to kit protocols for antibody-based detection of

US

cytokines.

Separation of Immune Cells from Stromal Tissues

AN

Ileum, jejunum and Peyer’s patches samples were removed from HBSS, placed in collagenase solution (2 mL/sample, Sigma Aldrich, Cat#C9891) and minced. Tissues were placed in an

M

incubator at 37°C for 25-30 minutes then removed and samples were triturated with 18 gauge

ED

needles on 3mL syringes. Tissues and collagenase solution were passed through a 40µm cell strainer, and rinsed with 10mL HBSS to deactivate collagenase. Visceral lymph nodes were

PT

dissociated and filtered through a 40 m cell strainer without the use of collagenase. All cell

CE

suspensions were centrifuged at 2000 rpm at 4°C for 5 minutes. Supernatant was poured off and samples were re-suspended in 400µL of FACS buffer (1% BSA, 0.1% sodium azide, and PBS)

AC

for immediate cell count (see below: Cell Counting) then temporarily stored.

Cell Counting – Total and Viable Tissue cell suspensions (see separation of immune cells from stromal tissues) were pipetted onto 96-well plates and mixed with trypan blue exclusion dye marker (ThermoFisher Scientific, Waltham, MA) at 1:1. Total and viable cell counts were evaluated on a Cellometer (Nexcelom, Lawrence, MA) using a pre-defined small immune cell program developed by Nexcelom. Immune cells were identified by diameter with a range from 4-20 microns.

ACCEPTED MANUSCRIPT

Flow Cytometry Between 5 x 105 and 1 x 106 cells from intestinal suspensions were plated per well. Cells were washed, spun and supernatant was removed and discarded. To prevent non-specific binding of antibodies cells were re-suspended in normal mouse block and briefly incubated. Primary

IP

T

antibodies were added at a 1:200 dilution in two different panels. Panel 1 included: anti-CD8

CR

(clone 53-6.7, cat. no. 13-0081-85, eBioscience), anti-CD4 (clone RM4-5, cat. no. 48-0042-82, eBioscience), anti-CD3 (clone 145-2c11, cat. no. 11-0031-82 eBioscience), and intracellular

US

Foxp3 (clone FJK-16a, cat. no. 12-5773-82, eBioscience) for T cell differentiation; anti-B220 (clone RA3-6B2, cat. no. 17-0452-82, eBioscience) for B cell identification; anti-NKG2D (clone

AN

CX5, cat. no. 13-5882-82, eBioscience) for natural killer cells. Panel 2) included: anti-CD11b (clone M1/70, cat. no. 48-0112-82, eBioscience), anti-CD11c (clone N418, cat. no. 417-0114-82,

M

eBioscience), anti-F4/80 (clone BM8, cat. no. 25-5931-82, eBioscience), and anti-MHCII (clone

ED

M5/114.15.2, cat. no. 17-5321-81, eBioscience) for antigen presenting cell differentiation. After incubation and washes with FACS buffer secondary antibody streptavidin-pacific orange (cat. no.

PT

532365, Invitrogen) was added at a dilution of 1:500. In addition, a permeabilization kit (cat. no.

CE

00-5521, eBioscience) was used in accordance with kit instructions for FoxP3 intracellular staining. Last, stained cells were fixed in 4% PFA, washed and stored in FACS buffer for analysis

AC

the following day. Flow cytometry of immune cell populations were analyzed using MoFlo Legacy Cell Analyzer and Sorter (Beckman Coulter, Indianapolis, IN). Data were analyzed using Summit software version 4.2 (Beckman Coulter, Indianapolis, IN).

Statistics Data are expressed as mean  standard error of the mean (SEM). Food intake, body weight, intestine tissue cell percent and viable counts were analyzed with a One-way ANOVA for significance values among individual data points. Post-hoc tests of individual groups were made

ACCEPTED MANUSCRIPT using LSD tests. Lymph node immune cell percent and viable number as well as adipose tissue cytokines were analyzed by T-test. For all experiments, differences among groups were considered statistically significant if p ≤ 0.05.

RESULTS

IP

T

Food intake and body weight.

CR

Cumulative kcal intake was significantly higher in HFD mice compared with Chow (Chow 387.54 kcals ± 9.19, HFD 555.9 kcals ± 8.75 ; p = 7.3E-5) following 7 weeks of intake. As a result

US

7 weeks of HFD intake significantly increased body mass (Chow 28.9 ± 0.67, WD 39.39 ± 0.77 ; p = 1.67E-9) compared with Chow controls. Consistent with this individual adipose tissue,

AN

epididymal, visceral, perirenal and inguinal white adipose tissue, and total adiposity (previous adipose tissue depots added together) were significantly increased in the HFD group (Table 1; p

Adipose Depot Cytokines

ED

M

≤ 0.05).

PT

IL-1β, IL-5 and IL-6 (Figure 1 A-C) concentrations were increased in the visceral adipose depots

CE

of HFD mice, both IL-5 (Figure 1B; p = 0.05) and -6 (Figure 1C; p = 0.003) were significantly increased compared with controls. IL-13, however, decreased in the visceral depot of HFD

AC

animals (Figure 1 D). In addition, KC GRO (a.k.a. chemokine (C-X-C motif) ligand 1 (CXCL1) and tumor necrosis factor α (TNFα) protein concentration was measured. KC GRO is thus far characterized to be increased during pro-inflammation and obesity and aids as a chemoattractant for neutrophils [41]. TNFα is a protein involved in systemic inflammation. Both KC GRO (Figure 1E; p = 0.018) and TNFα (Figure 1F; p = 0.019) were significantly increased in HFD mice compared with control.

Visceral Lymph Node Cell Number and Flow Cytometry

ACCEPTED MANUSCRIPT 7 weeks of HFD significantly increased the number of viable cells within the visceral lymph node (Figure 2A; p = 0.02). In general, HFD did not shift immune cell percent within the visceral lymph node, with the exception of CD4+Foxp3+ regulatory T cells (Tregs) immune cells that play a role in suppression of immune response. The percent of Tregs were significantly decreased in HFD mice compared with CHOW control (Figure 2B; p = 0.039). Absolute numbers of distinct immune

IP

T

cell subsets were determined by multiplying percent of representative cell populations by the total

CR

viable cell count. This data is represented in Figure 2C. HFD intake did not cause vast shifts in immune cell frequencies, but did cause multiple significant changes in viable visceral lymph node

US

immune cell numbers. Specifically, mice fed HFD had significantly higher numbers of F4/80+CD11b+ macrophages (p = 0.022), CD11b+CD11c+ dendritic cells (p = 0.017), CD3+CD4+

AN

helper T cells (p = 0.024), CD3+ general T cells (p = 0.02) and B220+ B cells (p = 0.002) within the visceral lymph node compared with CHOW control (Figure 2C). In opposition, CD4+Foxp3+

ED

M

regulatory T cells viable number was decreased in HFD mice (Figure 2C; p = 0.011).

Intestine Cell number and Flow Cytometry

PT

7 weeks of HFD did not alter total viable cell number in ileum, jejunum or Peyer’s patch samples

CE

(Supplemental Figure 1). A limitation of this study was the processing of whole intestinal sections. This method of collection interferes with the determination of viable immune cell populations

AC

because of the numerous different cells types located within the intestines that are a similar size to immune cells of interest. Hence, viable cell number for the intestines would not be reflective of the actual immune cell number in the intestine. Therefore, only percent frequency will be reported for the ileum, jejunum and Peyer’s patches. Immune cell frequency was evaluated in several distinct intestine regions because of the inherent anatomical and physiological differences between intestine segments (For review see:[30]). Differences in epithelial cell number, absorption capacity and numerous other factors can influence immune regulation. In general, changes in immune cell frequency where similar

ACCEPTED MANUSCRIPT between intestine regions. First, in the ileum HFD caused significant decreases (Figure 3A) in percent of CD4+Foxp3+ regulatory T (p = 0.011), NKG2D+ (p = 0.002) and NKG2D+CD4+ (P = 0.014) immune cells while significantly increasing percent of CD11c+MCHII+ dendritic cells (p = 0.016) compared with CHOW. Similarly, in the jejunum the percent of CD4+Foxp3+ regulatory T (p = 0.015) and NKG2D+CD4+ (P = 0.034) immune cells were significantly decreased and

IP

T

CD11c+MCHII+ dendritic cells (p = 0.008) frequency was significantly increased in HDF mice. Additionally, HFD significantly decreased the percent of jejunum CD3+CD4+ helper T cells

CR

(Figure 4A; p = 0.011) compared with CHOW. Last, Peyer’s patches located along the ileum and

US

jejunum (Figure 5) of HFD mice had significant decreases in percent of CD4+Foxp3+ regulatory T (p = 0.011), NKG2D+ (p = 0.002), NKG2D+CD4+ (P = 0.014) and CD11b+GR1+ myeloid

AN

suppressor (p = 0.042) immune cells. Similar to the ileum and jejunum CD11c+MCHII+ dendritic cells (p = 0.005) in Peyer’s patches were significantly increased in HFD mice compared with

ED

M

CHOW (Figure 5).

DISSCUSSION

PT

Obesity induces chronic low-grade inflammation. It is suggested that obesity associated

CE

inflammation is incited and exacerbated by excessive dysregulated adipose tissue deposition. Dysregulation of adipose tissue endocrine function, pro-inflammatory cytokines and deleterious

AC

cytokines are implicated in pathophysiology of numerous diseases. Visceral adipose tissue accumulation, in particular, is highly associated with adverse metabolic profiles [42]. Emerging evidence additionally supports a relation between dysregulated visceral adipose tissue and gut dysfunction [43]. Specifically, visceral adipose tissue pro-inflammation causes gastrointestinal leakiness [44, 45] and gut inflammation drives adipose tissue accumulation [46], both factors together exacerbate systemic pro-inflammation. Because the gastrointestinal tract and visceral adipose tissue commonly drain to visceral lymph node, we proposed to investigate the associated changes between the three intra-abdominal tissues during obesity. Here we demonstrated that

ACCEPTED MANUSCRIPT both the visceral adipose depot and the gastrointestinal tract likely influence immune cells enclosed within the visceral lymph node. This occurred by HFD-induced increases in visceral adipose tissue pro-inflammatory cytokines and a reduction in the tolerant milieu of the intestines needed to suppress unnecessary immune reaction to commensal bacteria and beneficial nutrients.

IP

T

Visceral adipose tissue accumulation is highly associated with metabolic disease [24, 25].

CR

Deleterious effects of excessive visceral depot expansion, in part, are due to increased production and release of pro-inflammatory cytokines [47] that circulate directly to the liver and systemically.

US

Cytokine release from adipose tissue originate from both adipocytes and immune cells contained within the depot in response to the changing milieu induced by HFD. Consistent with previous

AN

studies, we demonstrate that HFD-induces deleterious cytokine release from visceral adipose tissue. Il-1 plays a fundamental role in immune response. These molecules comprise a family

M

of pro-inflammatory cytokines that induce a complex network of additional pro-inflammatory

ED

cytokines/mediators via interaction with leukocytes. Specifically, IL-1b in adipose tissue is known as mononuclear cell factor and a lymphocyte activating factor that originates from

PT

macrophages and is activated by adipocyte-specific caspase-1 [48]. Specifically, release of IL-

CE

1b, in a situation such as obesity, enhances antigen-mediated expansion of pro-inflammatory cell types such as Th1 and Th17 T cells [49]. IL-1b also promotes the recruitment and retention

AC

of macrophages [50]. Similar to IL-1b, IL-6 is a fundamental regulator of T cells. Of the numerous immune-modulating cytokines increased in obesity, IL-6 dysregulation significantly contributes to pro-inflammation and metabolic dysregulation [51, 52]. Release of Il-6 causes numerous events to occur that allow for T cell survival. This includes promoting T cell development [53] and activation and tissue invasion [54, 55]. In addition, Il-6 permits effector T cells to overcome suppression by regulatory T cells [55, 56] while correspondingly inhibiting immune suppression T cells [57]. In opposition are interleukins associated with anti-inflammation that are typically decreased in obesity including IL-5 and IL-13. IL-5 plays a role in maintaining immune cell

ACCEPTED MANUSCRIPT populations that contribute to metabolic homeostasis including glucose tolerance, and protection against diet-induced obesity in adipose tissue [58, 59]. Similarly, IL-13 is also associated with conservation of glucose homeostasis [60]. In response to a pathogen TNFα is typically the first cytokine released [61]. Within tissues TNFα recruits cells towards pro-inflammatory stimuli by inducing vasodilation, which allows increased infiltration of immune cells such as lymphocytes,

IP

T

neutrophils and monocytes [62]. TNFα also induces the release of KC GRO (CXCL1) from immune cells such macrophages and neutrophils [63, 64], this further facilitates pro-inflammatory

CR

immune cell recruitment. Obesity increases both TNFα [65] and CXCL1 release [63]. Overall, our

US

data demonstrates that 7 weeks of HFD increases pro-inflammatory cytokines while decreasing immune-suppressing cells types. This subsequently creates a signal in adipose tissue that permits

AN

increased infiltration of pro-inflammatory cells types. We propose effluent from visceral adipose tissue into the visceral lymph node influence immune cells incased within.

M

The visceral lymph node, however, is not limited to visceral adipose depot effluent. Intestine

ED

health can also influence the visceral lymph node. The gastrointestinal (GI) tract is the first line of protection from antigens and immunomodulatory agents consumed from our diet or localized

PT

within our gut. Hence, it is likely that immune dysregulation within the gut can directly influence

CE

immune cells encased within the visceral lymph node. In the present study, we comprehensively investigated how HFD altered immune cell populations within the small intestines by comparing

AC

distinct intestinal regions with inherent anatomical and physiological differences. The jejunum and ileum as well as Peyer’s patches contained throughout these two regions were evaluated for differential response to HFD. Distinct intestine sections were evaluated to determine if regional differences in specialized epithelial cells, receptor types, antigen uptake or absorption capacity could disparately influence HFD outcomes. Although there were some minor differences between gut regions, most changes among the three sections evaluated were similar. First this includes HFD-induced decrease in regulatory T cells (Tregs: CD4+foxp3+) in all three intestine tissues. These regulatory cells are fundamental in maintaining homeostasis within the

ACCEPTED MANUSCRIPT gut as demonstrated in numerous inflammatory disease models (For review see: [66]). Generally, Tregs are upregulated during homeostatic conditions and consequently downregulated in order to elicit an immune response to pathogens or other inflammatory stimuli [67]. This is the first indication that HFD induced obesity is potentially driving a shift towards the pro-inflammatory environment associated with obesity. As previously discussed, this shift in Treg cells is well

IP

T

characterized within the ileum and jejunum in obesity as well as other inflammatory bowel disorders [66] but to the best of our knowledge, this is the first time this has been documented

CR

within Peyer’s patches in response to diet induced obesity. Second are the dendritic cells (DCs:

US

CD11c+MHCII+) which are antigen presenting cells. Within the intestines, dendritic cells function to extend cellular extensions between epithelial cells to sample pathogens or commensal factors

AN

of the intestinal lumen. Dendritic cell percent was increased in all three HFD intestinal samples. Research demonstrates that harmful pathogens can trigger dendritic cells to elicit a pro-

M

inflammatory cascade that includes 1.) both attracting and stimulating maturation and

ED

differentiation of B and T lymphocytes to mature into effector cells [68, 69], 2.) sustainability of recirculating T lymphocytes [70], and 3.) stimulation of antibody production from B cells by IL-12

PT

secretion [71]. We would speculate these changes in immune system milieu would lead to a shift

CE

in percent increase of effector immune cells that play a role in pro-inflammation, yet this did not occur within the small intestine. Hence, the third common change to occur among our three tissue

AC

regions was decreased NKG2D+ immune cells, in particular those that where NKG2D+CD4+. The role of CD4 T cells expressing the NKG2D receptor is best described in Crohn’s disease [7274]. NKG2D+CD4+ cells in the lamina propria of patients presenting with Crohn’s disease were associated with a pro-inflammatory cytokine profile [72] with a high expression of both IFN-y, TNFα and increased cytotoxic capability [72]. Taken together, this demonstrates that NKG2D+CD4+ cells may play an important role in shaping the pro-inflammatory profile of the small intestine during HFD induced obesity and inflammation. However, we observed a decrease in NKG2D+CD4+ cells. Similarly, but specific to the Jejunum, there was also a decrease in small

ACCEPTED MANUSCRIPT intestine CD3+CD4+ effector T cells, this again was contradictory to our prediction. Last, studies demonstrate that myeloid derived suppressor cells (MDSCs), characterized as CD11B+GR1+, induce intestinal tolerance by suppressing T-cell [75]. In Peyer’s patches only, HFD decreased MDSCs compared with CHOW. Taken together, in the intestines HFD induces release of suppressive immune cell types typically needed for toleration of commensal bacteria and variable

IP

T

lumen components. Release of this suppression caused a pro-inflammatory environment with an

CR

increase in antigen presenting dendritic cells that present to and activate effector cells. Hence, an increase in dendritic cell migration to the intestine increases antigen presentation and

US

consequently T and B cell response [76]. However, there was not a corresponding increase in the percent of effector cells within the intestine instead there was a decrease. We propose this

AN

decrease in immune cell frequency may be due to immune cell migration from the gut. Data supports that gastrointestinal immunity and systemic tolerance is predominately mediated

M

by the visceral lymph node [40]. Gastrointestinal tolerance is the ability of immune cells to resist

ED

an unnecessary pro-inflammatory response to harmless food antigens and commensal bacteria and remain in a homeostatic state (for review see: [77]). The visceral lymph node serves as a

PT

conduit that continuously surveys and monitors exposure of the small intestine to potentially

CE

harmful pathogens and metabolites [17, 18]. Within this lymph node immune cells can be recruited and activated to defend against damage, pathogens or impaired function [19]. A fundamental role

AC

of the visceral lymph node is to create tolerance to beneficial nutrients and commensal bacteria. This process is prompted by anti-inflammatory antigen presenting dendritic cells that constitutively traffic from the intestinal epithelium and Peyer’s patches to the visceral lymph node where T cell tolerization can occur [78-80]. Consequently, this creates a protective barrier within the lymph node, via enrichment in tolerant T cells, which prevents unnecessary systemic pro-inflammatory priming reactions [81-83]. HFD, however, releases immune tolerance leading to enhanced proinflammation. As we have demonstrated, HFD-induced obesity is associated with an increase in dendritic cells within the intestines; we propose these cells are primed for immune response and

ACCEPTED MANUSCRIPT not tolerance (pro-inflammatory dendritic cells). As such, we would expect an associated increase in dendritic activated immune cells that defend against antigen presented to them (effector cells). This effector immune response did not occur within the intestine. The visceral lymph node, however, swelled in size as a response to HFD intake. The increase in size was due to an influx/proliferation of immune cells. In general, there was not a change in percent distribution of

IP

T

immune cell type within the visceral lymph node, besides decreased percent of T regs, but there

CR

was an increase in dendritic cells, macrophages and effector T and B cell types. Previous studies demonstrate that immune cells migrate from PPs [84, 85] to lymphatics in the mesenteric lymph

US

nodes [85]. The movement of lymphocyte populations is described to occur in reaction to mobile antigen presenting cells, such as dendritic cells (DCs) [86, 87]. Upon antigen/pathogen

AN

presentation dendritic cells mature and release cytokines while migrating from PPs to the mesentery. The release of cytokines is described to attract recently activated T lymphocytes [86].

M

We propose the diet-induced increase in the percent of dendritic cells and release of immune

ED

tolerance in the intestine causes immune cell expansion in the visceral lymph node. Here the visceral lymph node functions as a primary barrier that, in the case of HFD-induced inflammation,

PT

attempts to prevent the spread of regional pathogens and inflammatory stimuli.

CE

Last, considerations to take into account include data supporting a reciprocal relation among adipose tissue and the immune system regulation. Though this link remains to be

AC

elucidate, research supports that adipocytes also respond to signals emanating from embedded lymph nodes and/or leaky lymphatic vessel. Indeed, visceral adipose tissue is most sensitive to lymph node alterations and best demonstrates the functional basis for the anatomical association between the two tissues [88]. Pond et al. characterized adipose tissue surrounding lymph nodes to play a fundamental role in lymph node enlargement and immune response [89] by providing fatty acids via lipolysis from triacylglycerol stores [90-92]. In addition, lymph node stimulation is proposed to facilitate adipogenesis, where mild or chronic inflammation of lymphatic tissue is

ACCEPTED MANUSCRIPT associated with adipose depot enlargement [93]. In support of this lymphatic vessel fluid is also demonstrated to cause adipocyte hypertrophy [94]. Our studies support and extend that adipose tissue is an active endocrine organ involved in immunological processes. In our model, adipose tissue accumulation occurs by excessive food intake. However, adipose tissue accumulation and associated pro-inflammatory cytokine release could also be due to intestinal alterations that cause

IP

T

increases in pro-inflammatory immune milieu in the visceral lymph node. Further studies are

CR

needed to elucidate relative contributions of lymph node and adipose tissue response in the progression of obesity-induced systemic inflammation. It is likely they both contribute and

US

exacerbate obesity-induced inflammation.

Taken together this data demonstrates that visceral adipose tissue and the gastrointestinal tract

AN

mutually influence immune cells enclosed within the visceral lymph node. During HFD-induced obesity, excessive adipose tissue deposition leads to a shift in deleterious immune cell types

M

subsequently leading to increased release of several cytokines. This effluent is taken up by the

ED

visceral lymph node. Within the visceral lymph node adipose derived pro-inflammatory cytokines aid in the expansion, survival and retention of pro-inflammatory T cells (For example Th1 and

PT

Th17) and macrophages. Gastrointestinal dysfunction also contributes to this diet-induce

CE

metabolic dysregulation. The gastrointestinal tract and visceral adipose tissue likely cause an additive pro-inflammatory response to HFD-induced obesity in the visceral lymph node.

AC

Gastrointestinal tolerance is the ability of immune cells to resist an unnecessary pro-inflammatory response to harmless food antigens and commensal bacteria. The visceral lymph node contributes to gastrointestinal tolerance while also protecting systemic immunity by providing a barrier to prevent spread of gastrointestinal pathogens. However, diet-induced obesity leads to an inhibition of gastrointestinal tolerance, which consequently promotes migration of immune reactive cells to the visceral lymph node in an attempt to stimulate an immune barrier to protect systemic immunity. We propose, however, that chronic effluent of pro-inflammatory cytokines from excessive adipose tissue expansion will eventually disrupt this process and gratuitously

ACCEPTED MANUSCRIPT exacerbate lymph node immune cell expansion. We predict in obesity, this can result in immune cell dysfunction, exhaustion and immunosuppression within the intra-abdominal cavity.

Conflict of interest

IP

T

The authors declare that they have no conflicts of interest.

CR

Acknowledgements

This work was supported by the National Institutes of Health [Grant Numbers P30 DK048520 and

AC

CE

PT

ED

M

AN

US

R03 DK099425]

ACCEPTED MANUSCRIPT REFERENCES

8. 9.

10.

11. 12. 13. 14.

15. 16. 17.

18. 19.

T

IP

CR

US

7.

AN

6.

M

5.

ED

4.

PT

3.

CE

2.

Lamon-Fava, S., P.W. Wilson, and E.J. Schaefer, Impact of body mass index on coronary heart disease risk factors in men and women. The Framingham Offspring Study. Arterioscler Thromb Vasc Biol, 1996. 16(12): p. 1509-15. Miyake, T., et al., Body mass index is the most useful predictive factor for the onset of nonalcoholic fatty liver disease: a community-based retrospective longitudinal cohort study. J Gastroenterol, 2013. 48(3): p. 413-22. Sanada, H., et al., High body mass index is an important risk factor for the development of type 2 diabetes. Intern Med, 2012. 51(14): p. 1821-6. O'Neill, S. and L. O'Driscoll, Metabolic syndrome: a closer look at the growing epidemic and its associated pathologies. Obes Rev, 2015. 16(1): p. 1-12. Bjorntorp, P., Metabolic implications of body fat distribution. Diabetes Care, 1991. 14(12): p. 1132-43. De Pergola, G. and F. Silvestris, Obesity as a major risk factor for cancer. J Obes, 2013. 2013: p. 291546. Bilecik, N.A., et al., Prevalence of metabolic syndrome in women with rheumatoid arthritis and effective factors. Int J Clin Exp Med, 2014. 7(8): p. 2258-65. Lee, C.G., et al., Visceral abdominal obesity is associated with an increased risk of irritable bowel syndrome. Am J Gastroenterol, 2015. 110(2): p. 310-9. Thijssen, E., A. van Caam, and P.M. van der Kraan, Obesity and osteoarthritis, more than just wear and tear: pivotal roles for inflamed adipose tissue and dyslipidaemia in obesityinduced osteoarthritis. Rheumatology (Oxford), 2014. Guarner, V. and M.E. Rubio-Ruiz, Low-grade systemic inflammation connects aging, metabolic syndrome and cardiovascular disease. Interdiscip Top Gerontol, 2015. 40: p. 99-106. Esser, N., et al., Inflammation as a link between obesity, metabolic syndrome and type 2 diabetes. Diabetes Res Clin Pract, 2014. 105(2): p. 141-50. Mraz, M. and M. Haluzik, The role of adipose tissue immune cells in obesity and low-grade inflammation. J Endocrinol, 2014. 222(3): p. R113-27. Kissebah, A.H. and G.R. Krakower, Regional adiposity and morbidity. Physiol Rev, 1994. 74(4): p. 761-811. Vague, J., The degree of masculine differentiation of obesities: a factor determining predisposition to diabetes, atherosclerosis, gout, and uric calculous disease. Am J Clin Nutr, 1956. 4(1): p. 20-34. Bjorntorp, P., "Portal" adipose tissue as a generator of risk factors for cardiovascular disease and diabetes. Arteriosclerosis, 1990. 10(4): p. 493-6. Pond, C.M. and C.A. Mattacks, Interactions between adipose tissue around lymph nodes and lymphoid cells in vitro. J Lipid Res, 1995. 36(10): p. 2219-31. Arngrim, N., et al., Reduced adipose tissue lymphatic drainage of macromolecules in obese subjects: a possible link between obesity and local tissue inflammation? Int J Obes (Lond), 2013. 37(5): p. 748-50. Weitman, E.S., et al., Obesity impairs lymphatic fluid transport and dendritic cell migration to lymph nodes. PLoS One, 2013. 8(8): p. e70703. von der Weid, P.Y. and K.J. Rainey, Review article: lymphatic system and associated adipose tissue in the development of inflammatory bowel disease. Aliment Pharmacol Ther, 2010. 32(6): p. 697-711.

AC

1.

ACCEPTED MANUSCRIPT

28. 29. 30. 31. 32. 33.

34.

35. 36. 37. 38.

39.

T

IP

CR

US

27.

AN

26.

M

25.

ED

24.

PT

23.

CE

21. 22.

Pond, C.M. and C.A. Mattacks, THE ACTIVATION OF THE ADIPOSE TISSUE ASSOCIATED WITH LYMPH NODES DURING THE EARLY STAGES OF AN IMMUNE RESPONSE. Cytokine, 2002. 17(3): p. 131-139. Yang, H., et al., Obesity accelerates thymic aging. Blood, 2009. 114(18): p. 3803-3812. Kim, Y.-H., et al., Diet-induced obesity dramatically reduces the efficacy of a 2009 pandemic H1N1 vaccine in a mouse model. Journal of Infectious Diseases, 2011. 205(2): p. 244-251. Karlsson, E.A. and M.A. Beck, The burden of obesity on infectious disease. Experimental biology and medicine, 2010. 235(12): p. 1412-1424. Nielsen, S., et al., Splanchnic lipolysis in human obesity. J Clin Invest, 2004. 113(11): p. 1582-8. van der Poorten, D., et al., Visceral fat: a key mediator of steatohepatitis in metabolic liver disease. Hepatology, 2008. 48(2): p. 449-57. de Wit, N.J., et al., The role of the small intestine in the development of dietary fat-induced obesity and insulin resistance in C57BL/6J mice. BMC Med Genomics, 2008. 1: p. 14. Mowat, A.M., Anatomical basis of tolerance and immunity to intestinal antigens. Nat Rev Immunol, 2003. 3(4): p. 331-41. Helander, H.F. and L. Fandriks, Surface area of the digestive tract - revisited. Scand J Gastroenterol, 2014. 49(6): p. 681-9. Vighi, G., et al., Allergy and the gastrointestinal system. Clin Exp Immunol, 2008. 153 Suppl 1: p. 3-6. Mowat, A.M. and W.W. Agace, Regional specialization within the intestinal immune system. Nat Rev Immunol, 2014. 14(10): p. 667-85. Kunisawa, J., Y. Kurashima, and H. Kiyono, Gut-associated lymphoid tissues for the development of oral vaccines. Advanced Drug Delivery Reviews, 2012. 64(6): p. 523-530. Wershil, B.K. and G.T. Furuta, 4. Gastrointestinal mucosal immunity. J Allergy Clin Immunol, 2008. 121(2 Suppl): p. S380-3; quiz S415. Ding, S., et al., High-fat diet: bacteria interactions promote intestinal inflammation which precedes and correlates with obesity and insulin resistance in mouse. PLoS One, 2010. 5(8): p. e12191. Novotny Nunez, I., et al., Lactobacillus casei CRL 431 administration decreases inflammatory cytokines in a diet-induced obese mouse model. Nutrition, 2015. 31(7-8): p. 1000-7. Yoshida, H., et al., Fatty acids enhance GRO/CINC-1 and interleukin-6 production in rat intestinal epithelial cells. J Nutr, 2001. 131(11): p. 2943-50. Kvietys, P.R., et al., Jejunal mucosal injury and restitution: role of hydrolytic products of food digestion. Am J Physiol, 1991. 261(3 Pt 1): p. G384-91. Miura, S., et al., Modulation of intestinal immune system by dietary fat intake: relevance to Crohn's disease. J Gastroenterol Hepatol, 1998. 13(12): p. 1183-90. Lam, Y.Y., et al., Increased gut permeability and microbiota change associate with mesenteric fat inflammation and metabolic dysfunction in diet-induced obese mice. PLoS One, 2012. 7(3): p. e34233. Lam, Y.Y., et al., Effects of dietary fat profile on gut permeability and microbiota and their relationships with metabolic changes in mice. Obesity (Silver Spring), 2015. 23(7): p. 1429-39.

AC

20.

ACCEPTED MANUSCRIPT

48. 49. 50. 51.

52.

53.

54. 55. 56.

57. 58.

T

IP

CR

47.

US

46.

AN

45.

M

44.

ED

43.

PT

42.

CE

41.

Macpherson, A.J. and K. Smith, Mesenteric lymph nodes at the center of immune anatomy. J Exp Med, 2006. 203(3): p. 497-500. Oliveira, M.C., et al., Acute and sustained inflammation and metabolic dysfunction induced by high refined carbohydrate-containing diet in mice. Obesity (Silver Spring), 2013. 21(9): p. E396-406. Porter, S.A., et al., Abdominal subcutaneous adipose tissue: a protective fat depot? Diabetes Care, 2009. 32(6): p. 1068-75. Lam, Y.Y., et al., Role of the gut in visceral fat inflammation and metabolic disorders. Obesity (Silver Spring), 2011. 19(11): p. 2113-20. Cani, P.D., et al., Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes, 2008. 57(6): p. 1470-81. Everard, A., et al., Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proc Natl Acad Sci U S A, 2013. 110(22): p. 9066-71. Gambero, A., et al., Mesenteric adipose tissue alterations resulting from experimental reactivated colitis. Inflamm Bowel Dis, 2007. 13(11): p. 1357-64. Booth, A., A. Magnuson, and M. Foster, Detrimental and protective fat: body fat distribution and its relation to metabolic disease. Horm Mol Biol Clin Investig, 2014. 17(1): p. 13-27. Stienstra, R., et al., The inflammasome-mediated caspase-1 activation controls adipocyte differentiation and insulin sensitivity. Cell Metab, 2010. 12(6): p. 593-605. Ben-Sasson, S.Z., et al., IL-1 acts directly on CD4 T cells to enhance their antigen-driven expansion and differentiation. Proc Natl Acad Sci U S A, 2009. 106(17): p. 7119-24. Rider, P., et al., IL-1alpha and IL-1beta recruit different myeloid cells and promote different stages of sterile inflammation. J Immunol, 2011. 187(9): p. 4835-43. Pickup, J.C., et al., NIDDM as a disease of the innate immune system: association of acutephase reactants and interleukin-6 with metabolic syndrome X. Diabetologia, 1997. 40(11): p. 1286-92. Kern, P.A., et al., Adipose tissue tumor necrosis factor and interleukin-6 expression in human obesity and insulin resistance. Am J Physiol Endocrinol Metab, 2001. 280(5): p. E745-51. Atreya, R., et al., Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo. Nat Med, 2000. 6(5): p. 583-8. McLoughlin, R.M., et al., IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation. Proc Natl Acad Sci U S A, 2005. 102(27): p. 9589-94. Nish, S.A., et al., T cell-intrinsic role of IL-6 signaling in primary and memory responses. Elife, 2014. 3: p. e01949. Yoshida, H., et al., Anti-IL-6 receptor antibody suppressed T cell activation by inhibiting IL-2 production and inducing regulatory T cells. Eur J Pharmacol, 2010. 634(1-3): p. 17883. Bettelli, E., et al., Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature, 2006. 441(7090): p. 235-8. Wu, D., et al., Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis. Science, 2011. 332(6026): p. 243-7.

AC

40.

ACCEPTED MANUSCRIPT

67. 68. 69. 70.

71. 72.

73.

74. 75. 76.

77.

T

IP

CR

66.

US

65.

AN

64.

M

63.

ED

62.

PT

61.

CE

60.

Molofsky, A.B., et al., Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages. J Exp Med, 2013. 210(3): p. 535-49. Jiang, L.Q., et al., Autocrine role of interleukin-13 on skeletal muscle glucose metabolism in type 2 diabetic patients involves microRNA let-7. Am J Physiol Endocrinol Metab, 2013. 305(11): p. E1359-66. Beutler, B.A., The role of tumor necrosis factor in health and disease. J Rheumatol Suppl, 1999. 57: p. 16-21. Arango Duque, G. and A. Descoteaux, Macrophage cytokines: involvement in immunity and infectious diseases. Front Immunol, 2014. 5: p. 491. Nunemaker, C.S., et al., Increased serum CXCL1 and CXCL5 are linked to obesity, hyperglycemia, and impaired islet function. J Endocrinol, 2014. 222(2): p. 267-76. Griffin, G.K., et al., IL-17 and TNF-alpha sustain neutrophil recruitment during inflammation through synergistic effects on endothelial activation. J Immunol, 2012. 188(12): p. 6287-99. Weisberg, S.P., et al., Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest, 2003. 112(12): p. 1796-808. Sun, M., et al., Regulatory immune cells in regulation of intestinal inflammatory response to microbiota. Mucosal Immunol, 2015. 8(5): p. 969-978. Josefowicz, S.Z., et al., Extrathymically generated regulatory T cells control mucosal TH2 inflammation. Nature, 2012. 482(7385): p. 395-9. Adema, G.J., et al., A dendritic-cell-derived C-C chemokine that preferentially attracts naive T cells. Nature, 1997. 387(6634): p. 713-7. Ghiringhelli, F., et al., Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors. Nat Med, 2009. 15(10): p. 1170-8. Brocker, T., Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expressing dendritic cells. J Exp Med, 1997. 186(8): p. 1223-32. Dubois, B., et al., Dendritic cells enhance growth and differentiation of CD40-activated B lymphocytes. J Exp Med, 1997. 185(5): p. 941-51. Allez, M., et al., CD4+NKG2D+ T cells in Crohn's disease mediate inflammatory and cytotoxic responses through MICA interactions. Gastroenterology, 2007. 132(7): p. 234658. Pariente, B., et al., Activation of the receptor NKG2D leads to production of Th17 cytokines in CD4+ T cells of patients with Crohn's disease. Gastroenterology, 2011. 141(1): p. 21726, 226 e1-2. Camus, M., et al., Oligoclonal expansions of mucosal T cells in Crohn's disease predominate in NKG2D-expressing CD4 T cells. Mucosal Immunol, 2014. 7(2): p. 325-34. Haile, L.A., et al., Myeloid-derived suppressor cells in inflammatory bowel disease: a new immunoregulatory pathway. Gastroenterology, 2008. 135(3): p. 871-81, 881 e1-5. Kerjaschki, D., et al., Lymphatic neoangiogenesis in human kidney transplants is associated with immunologically active lymphocytic infiltrates. J Am Soc Nephrol, 2004. 15(3): p. 603-12. Maloy, K.J. and F. Powrie, Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature, 2011. 474(7351): p. 298-306.

AC

59.

ACCEPTED MANUSCRIPT

85.

86. 87. 88. 89. 90. 91.

92.

93.

94.

T

IP

CR

US

84.

AN

83.

M

82.

ED

81.

PT

80.

CE

79.

Huang, F.P., et al., A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J Exp Med, 2000. 191(3): p. 435-44. Worthington, J.J., et al., Intestinal dendritic cells specialize to activate transforming growth factor-beta and induce Foxp3+ regulatory T cells via integrin alphavbeta8. Gastroenterology, 2011. 141(5): p. 1802-12. Sun, J.B., C. Czerkinsky, and J. Holmgren, Sublingual 'oral tolerance' induction with antigen conjugated to cholera toxin B subunit generates regulatory T cells that induce apoptosis and depletion of effector T cells. Scand J Immunol, 2007. 66(2-3): p. 278-86. Hadis, U., et al., Intestinal tolerance requires gut homing and expansion of FoxP3+ regulatory T cells in the lamina propria. Immunity, 2011. 34(2): p. 237-46. Worbs, T., et al., Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells. J Exp Med, 2006. 203(3): p. 519-27. Schulz, O., et al., Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions. J Exp Med, 2009. 206(13): p. 310114. von Andrian, U.H. and T.R. Mempel, Homing and cellular traffic in lymph nodes. Nat Rev Immunol, 2003. 3(11): p. 867-78. Koboziev, I., F. Karlsson, and M.B. Grisham, Gut-associated lymphoid tissue, T cell trafficking, and chronic intestinal inflammation. Ann N Y Acad Sci, 2010. 1207 Suppl 1: p. E86-93. Sallusto, F. and A. Lanzavecchia, Mobilizing dendritic cells for tolerance, priming, and chronic inflammation. J Exp Med, 1999. 189(4): p. 611-4. Cella, M., F. Sallusto, and A. Lanzavecchia, Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol, 1997. 9(1): p. 10-6. Pond, C.M. and C.A. Mattacks, The activation of the adipose tissue associated with lymph nodes during the early stages of an immune response. Cytokine, 2002. 17(3): p. 131-9. Pond, C.M. and C.A. Mattacks, The source of fatty acids incorporated into proliferating lymphoid cells in immune-stimulated lymph nodes. Br J Nutr, 2003. 89(3): p. 375-83. Pond, C.M. and C.A. Mattacks, In vivo evidence for the involvement of the adipose tissue surrounding lymph nodes in immune responses. Immunol Lett, 1998. 63(3): p. 159-67. Mattacks, C.A. and C.M. Pond, Interactions of noradrenalin and tumour necrosis factor alpha, interleukin 4 and interleukin 6 in the control of lipolysis from adipocytes around lymph nodes. Cytokine, 1999. 11(5): p. 334-46. Mattacks, C.A., D. Sadler, and C.M. Pond, Site-specific differences in the action of NRTI drugs on adipose tissue incubated in vitro with lymphoid cells, and their interaction with dietary lipids. Comp Biochem Physiol C Toxicol Pharmacol, 2003. 135(1): p. 11-29. Mattacks, C.A., D. Sadler, and C.M. Pond, The cellular structure and lipid/protein composition of adipose tissue surrounding chronically stimulated lymph nodes in rats. J Anat, 2003. 202(6): p. 551-61. Medina, M. and D.P. Orgill, Discussion: regulation of adipogenesis by lymphatic fluid stasis: part I. Adipogenesis, fibrosis, and inflammation. Plast Reconstr Surg, 2012. 129(4): p. 835-7.

AC

78.

ACCEPTED MANUSCRIPT Table 1. Total and individual adipose tissue weight depots in grams. WD feeding magnifies total adipose, and expansion is.

7 week 0.63 0.09 0.08 0.15 0.20

0.011 0.003 0.001 0.030 0.010

T

5.34 1.87 1.25 1.08 1.41

p value

CR

Total Epididymal Visceral Perirenal Inguinal

HFD ± ± ± ± ±

IP

Chow 2.74 ± 0.48 1.10 ± 0.16 0.32 ± 0.02 0.59 ± 0.11 0.61 ± 0.14

AC

CE

PT

ED

M

AN

US

Table 1: Total and individual adipose tissue weight depots in grams. Total adiposity is significantly greater in HFD fed mice compared with CHOW (p= .011). All individual depot weights are significantly greater in HFD fed mice compared with CHOW (≤.05).

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

AC

CE

Figure 1: Visceral adipose tissue cytokine concentrations (pg/ml). Compared with CHOW, HFD increased the concentration of pro-inflammatory interleukins IL-1b A.) and IL-6 (* = 0.003) C.). The anti-inflammatory IL-5 B.) was also significantly increased (* = 0.05) whereas the other IL-13 was decreased D.). Additionally pro-inflammatory cytokines that were significantly increased include KC GRO (* = 0.018) E.) and TNFα (* = 0.019) F.).

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

Figure 2: Visceral lymph node immune cell number and frequency. A.) Total visceral lymph node viable cells number - HFD significantly increased total number of immunes cells within the visceral lymph node compared with CHOW (* = 0.02). B.) Percent frequencies (%) of individual immune cell populations – CD4+FoxP3+ cells (regulatory T) were the only immune cells to be significantly decreased in HFD fed mice compared with CHOW (* = 0.039). The inset represents scatter plots of the significant population enclosed within the circle. C.) Viable immune cells for distinct populations - F4/80+CD11b+macrophages (* = 0.022), CD11b+CD11c+ dendritic cells (* = 0.017), CD3+CD4+helper T cells (* = 0.024), CD3+ pan T cell (* = 0.02), and B220+ B cells (* = 0.002) were increased in the visceral lymph node of HFD fed mice compared with CHOW. CD4+FoxP3+ regulatory T cells, however, were decreased in HFD mice (** = 0.011)

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

AC

Figure 3: Immune cell frequency (%) for specific populations within the ileum. A.) CD4+FoxP3+ regulatory T (* = 0.011), NKG2D+ (* = 0.002) and NKG2D+CD4+ (* = 0.014) immune cells were significantly decreased in HFD fed mice compared with CHOW, whereas CD11c+MHCII+dendritic cells (* = 0.014) were significantly increased. B.) Representative scatter plots of immune cell frequency for populations significantly altered by HFD. Circles indicate double positive populations while the square represents all cells positive for a single marker, NKG2D.

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

AC

CE

PT

Figure 4: Immune cell frequency (%) for specific populations within the jejunum. A.) CD4+FoxP3+ regulatory T (* = 0.015), NKG2D+CD4+ (* = 0.034), and CD3+CD4+ helper T cells (* = 0.011) were significantly decreased in HFD fed mice compared with CHOW, whereas CD11c+MHCII+dendritic cells (* = 0.008) were significantly increased. B.) Representative scatter plots of immune cell frequency for populations significantly altered by HFD. All populations are double positive and indicated by circles.

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

AC

Figure 5: Immune cell frequency (%) for specific populations within the Peyer’s patches collected along the Ileum and jejunum. A.) Significant decreases occurred in CD4+FoxP3+ regulatory T (* = 0.011), NKG2D+ (*= 0.002), NKG2D+CD4+ (p= 0.014) and CD11b+Gr1+ Myeloid suppressor (* = 0.042) immune cells of HFD mice compared with CHOW. The frequency of CD11b+MHCII+ dendritic cells, however, were significantly increased in HFD mice (* = 0.005). B.) Representative scatter plots of immune cell frequency for populations significantly altered by HFD. The circles indicate double positive populations while the square indicates all cells positive for a single marker NKG2D.

ACCEPTED MANUSCRIPT Highlights

T IP CR US AN M ED PT



CE



Visceral adipose tissue and the gastrointestinal tract mutually influence immune cells of the visceral lymph node. Within the visceral lymph node adipose cytokine effluent aids in the expansion, survival and retention of pro-inflammatory T cells. HFD inhibits gastrointestinal tolerance, which promotes migration of immune reactive cells to the visceral lymph node.

AC

